Metals play an important role in biological systems in regulating enzyme activity, protein structure, and cellular signalling. Metals can also have a deleterious effect when present in excess of bodily requirements or capacity to excrete. A large number of natural and synthetic materials are known to selectively or non-selectively bind to or chelate metals. Ion chelators are commonly used in solution for in vivo control of ionic concentrations and detoxification of excess metals, and as in vitro buffers and optical indicators of ionic transients.
In addition, metal chelators immobilized on a carrier have been described for use in 1) removal or selective concentration of polyvalent metal ions from aqueous solutions and from metal binding sites of proteins; 2) attachment to antibodies and conjugation with gamma emitters such as Gd.sup.3+ and In.sup.3+ for use in delivering deadly doses of radiation to tumor cells (J. MED. CHEM. 17, 1304 (1974); J. MED. CHEM. 22, 1019 (1979)); 3) time resolved fluorescence immunoassays that usually use Tb.sup.3+ or Eu.sup.3+ complexes (ANALYT. BIOCHEM. 137, 335 (1984)); 4) radioimmunoassays; 5) gamma camera and NMR imaging (SCIENCE 209, 295 (1980); PROC. NAT'L ACAD. SCI. 83, 4277 (1986)); and 6) structural studies of the microenvironment and dynamic properties of proteins, membranes and nucleic acids.
Virtually all of the chelator compounds used for these applications have been derivatives of iminodiacetic acid (IDA as in Chelex.TM. ion-selective resins) or related amine aliphatic compounds such as ethylenediamine tetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA) or of ethylene glycol-bis(.beta.-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). Several reactive derivatives of EDTA and DTPA are known. See e.g. Meares, et al., ANALYT. BIOCHEM. 142, 68 (1984); Cummins, et al., BIOCONJUGATE CHEM. 2, 180 (1991). Of the IDA-like indicators, EGTA has the highest selectivity and affinity for Ca.sup.2+ versus Mg.sup.2+ and other polyvalent metals. The IDA, EDTA, DTPA and EGTA chelators with aliphatic amine groups, however, are all protonated at physiological pH (about pH 7), which significantly decreases their affinity. The need exists for a polymolecular carrier incorporating a highly selective ion chelator that does not show a significant decrease in affinity at physiological pH.
Among the highest affinity and most selective of the chelators, especially for Ca.sup.2+, have been derivatives of 1,2-bis-(2-aminophenoxyethane)-N,N,N',N'-tetraacetic acid (BAPTA) originally described by Tsien, BIOCHEM. 19, 2396 (1980). BAPTA has a high affinity for Ca.sup.2+ and certain other polyvalent metals such as the lanthanides Tb.sup.3+, Eu.sup.3+, Gd.sup.3+, Ln.sup.3+, and Zn.sup.2+, Pb.sup.2+, Mn.sup.2+, Cd.sup.2+ and Ba.sup.2+. Unlike the aliphatic amine chelators, the metal affinity of BAPTA chelators is about the same at pH 7 as above pH 7. In addition, the binding affinity of BAPTA for polyvalent metals, is more selective than the IDA-like chelators. The dissociation constant of Ca.sup.2 +-BAPTA is reported to be 107 nM whereas the dissociation constant of Mg.sup.2 +-BAPTA is reported to be 17 mM. This approximately 10.sup.6 difference in affinity for Ca.sup.2+ versus Mg.sup.2+ permits, for instance, the selective removal or buffering of Ca.sup.2+ in the presence of high concentrations of Mg.sup.2+. The affinity of BAPTA-derived chelators can be slightly increased by addition of electron donating groups and decreased by addition of electron withdrawing groups (Tsien, supra; CELL CALCIUM 10, 491 (1989)).
Scientists have taken advantage of the selectivity and affinity of BAPTA to develop Ca.sup.2+ ion selective fluorescent indicators that incorporate the basic BAPTA structure, including quin-2, fura-2 and indo-1 (U.S. Pat. No. 4,603,209 to Tsien, et al. (1986) (`209 patent); fluo-3 and rhod-2 (U.S. Pat. No. 5,049,673 to Tsien, et al. 1991) (`673 patent); and FURA RED.TM. (Molecular Probes, Inc., Eugene, Oreg., trademark for 1-[6-amino-2-(5-oxo-2-thioxo-4-thiazolidinylidene) methyl-5-benzofuranyloxy]-2-(2,2-amino-5'-methyl-phenoxy) ethane N,N, N', N'-tetraacetic acid and the tetra acetyloxymethyl ester thereof, U.S. Pat. No. 4,849,362 to DeMarinis, et al. (1989)). The structure of all these fluorescent indicators incorporates the aromatic BAPTA ring into a conjugated heterocyclic system through a trans-ethylenic bond which is either fixed (e.g. fura-2 and FURA RED) or rotating (e.g. fluo-3 and indo-1). Additional fluorescent indicators for Ca.sup.2+ have been described by Tsien (Intracellular Measurements of Ion Activities, ANN. REV. BIOPHYS. BIOENG. 12, 91 (1983)), however these all have limitations in fluorescence response or other properties and do not involve the formation of reactive intermediates such as those described in this invention. All these ion selective indicators exhibit a change in optical properties upon binding Ca.sup.2+ that can be used to determine changes in the levels of intracellular Ca.sup.2+.
Although numerous publications have described the use of BAPTA derivatives as soluble indicators and buffers to either measure or control ion concentrations, BAPTA derivatives with one or two reactive groups used to attach the BAPTA derivative to a polymolecular assembly while preserving the high Ca.sup.2+ affinity and Ca.sup.2+ versus Mg.sup.2+ selectivity of BAPTA have not previously been described. The `209 to Tsien, et al. discloses an amino-, aminomethyl-, and aminoethyl-aldehyde BAPTA intermediate used to synthesize fused heterocyclic aromatic indicators with a trans-ethylenic linkage between BAPTA and the fluorophore, but the `209 patent does not describe a reactive BAPTA derivative that can be used to conjugate BAPTA to a carrier and/or fluorophore with a linkage that contains more than one sigma bond in a row. The same linkage that allows for attachment of the chelator molecule to a wide range of polymolecular materials, also allows for attachment of the chelators to oxygen-containing heterocyclic fluorophores, to form novel and improved indicators.
Some of the indicator compounds formed in this way have an excitation wavelength that extends into the red range giving an emission well removed from any cellular background fluorescence and lowering light damage to cellular structures. The shorter wavelength light needed to excite fura-2 and indo-1 (340-360 nm) has been shown to damage cellular structures. The development of fluo-3 and rhod-2, with peak excitations wavelengths at 505 nm and 550 nm respectively, lowered UV induced cell damage and interference from cellular background fluorescence, but not as effectively as the most effective of the new compounds. Furthermore, the complex synthetic methods used to form the prior art indicators with the trans-ethylenic linkage limits the number of useful compounds that can be made in this way. The relatively easier conjugation of the reactive chelators of this invention increases the number of indicator compounds that can be made readily available.